The present invention relates to a quaternary ammonium salt which has CCR5 antagonistic activity and which is used for the treatment or prevention of infectious disease of HIV, etc.
Recently, HIV (human immunodeficiency virus) protease inhibitors are developed for method of the treatment of AIDS (acquired immunological deficient syndrome) and use of the protease inhibitors in combination with conventional two HIV reverse transcriptase inhibitors provides with a further progress of the treatment of AIDS. However, these drugs and their combination use are not sufficient for the eradication of AIDS, and development of new anti-AIDS drugs having different activity and mechanism are sought for.
As a receptor from which HIV invades to a target cell, CD4 is so far known, and recently CCR5 as a second receptor of macrophage-tropic HIV and CXCR4 as a second receptor of T cell-tropic HIV, each of which is G protein-coupled chemokine receptor having seven transmembrane domains, are respectively found out. These chemokine receptors are thought to play an essential role in establishment and spread of HIV infection. In fact, it is reported that a person who is resistant to HIV infection in spite of several exposures retains mutation of homo deletion of CCR5 gene. Therefore, a CCR5 antagonist is expected to be a new anti-HIV drug. However, so far, there has been no report that a CCR5 antagonist is developed as a therapeutic agent of AIDS.
The present invention is to provide a quaternary ammonium salt having CCR5 antagonistic activity and less toxicity; and a composition for antagonizing CCR5 (a drug for the treatment or prevention of infectious disease of HIV (in particular, AIDS), etc.) comprising said quaternary ammonium salt.
The present inventors diligently made extensive studies on compounds having CCR5 antagonistic activity and less toxicity, as a result, they found that a quaternary ammonium salt of the following formula (I) [hereinafter, referred to as Compound (I)] unexpectedly possesses potent CCR5 antagonistic activity and clinically desirable pharmaceutical effect (e.g. remarkable inhibition of HIV infection to human peripheral mononuclear cells, etc.) and also that Compound (I) has superior solubility, physicochemical properties (stability, anti-coloring effect, etc.), etc. and is useful for applying as an injection. Based on the finding, the present invention was accomplished.
More specifically, the present invention relates to
(1) a compound of the formula: 
wherein R1 is an optionally substituted phenyl or an optionally substituted thienyl; Y is xe2x80x94CH2xe2x80x94, xe2x80x94Sxe2x80x94 or xe2x80x94Oxe2x80x94; and R2, R3 and R4 are independently an optionally substituted aliphatic hydrocarbon group or an optionally substituted alicyclic heterocyclic ring group; or a pro-drug thereof;
(2) a compound of the above (1), wherein R2 and R3 are independently an optionally substituted acyclic hydrocarbon group;
(3) a compound of the above (1), wherein R2 and R3 are independently an optionally substituted alkyl group;
(4) a compound of the above (1), wherein R4is an optionally substituted alicyclic hydrocarbon group or an optionally substituted alicyclic heterocyclic ring group;
(5) a compound of the above (4), wherein the alicyclic hydrocarbon group is cycloalkyl;
(6) a compound of the above (4), wherein the alicyclic hydrocarbon group is cyclohexyl;
(7) a compound of the above (4), wherein the alicyclic heterocyclic ring group is a saturated alicyclic heterocyclic ring group;
(8) a compound of the above (4), wherein the alicyclic heterocyclic ring group is tetrahydropyranyl, tetrahydrothiopyranyl or piperidyl;
(9) N,N-Dimethyl-N-(4-(((2-(4-methylphenyl)-6,7-dihydro-5H-benzocyclohepten-8-yl)carbonyl)amino)benzyl)-N-(4-tetrahydropyranyl)ammonium chloride;
(10) N,N-Dimethyl-N-(((7-(4-methylphenyl)-2,3-dihydro-1-benzoxepin-4-yl)carbonyl)amino)benzyl)-N-(4-oxocyclohexyl)ammonium chloride;
(11) N-(4-(((7-(4-Ethoxyphenyl)-2,3-dihydro-1-benzoxepin-4-yl)carbonyl)amino)benzyl)-N,N-dimethyl-N-(4-tetrahydropyranyl)ammonium chloride;
(12) a pharmaceutical composition which comprises a compound of the above (1);
(13) a pharmaceutical composition for antagonizing CCR5 which comprises a compound of the above (9);
(14) a composition of the above (13), which is for the treatment or prevention of infectious disease of HIV;
(15) a composition of the above (13), which is for the treatment or prevention of AIDS;
(16) a composition of the above (13), which is for the prevention of the progression of AIDS;
(17) a composition of the above (13), which is used in combination with a protease inhibitor and/or a reverse transcriptases inhibitor;
(18) a composition of the above (17), wherein the reverse transcriptase inhibitor is zidovudine, didanosine, zalcitabine, lamivudine, stavudine, nevirapine, delavirdine, efavirenz 0r abacavir;
(19) a composition of the above (17), wherein the protease inhibitor is saquinavir, ritonavir, indinavir or nelfinavir;
(20) use of the compound of the above (9) in combination with a protease inhibitor and/or a reverse transcriptase inhibitor for the treatment or prevention of infectious disease of HIV; etc.
In the above formula (I), example of the xe2x80x9csubstituentsxe2x80x9d which the xe2x80x9cphenyl groupxe2x80x9d and the xe2x80x9cthienyl groupxe2x80x9d of the xe2x80x9coptionally substituted phenyl groupxe2x80x9d and xe2x80x9coptionally substituted thienyl groupxe2x80x9d represented by R1 may have include halogen atom, nitro, cyano, an optionally substituted alkyl, an optionally substituted cycloalkyl, an optionally substituted hydroxy group, an optionally substituted thiol group, an optionally substituted amino group, an optionally substituted acyl, an optionally esterified carboxyl group, an optionally substituted aromatic group, etc.
Examples of the halogen as the substituents for R1 include fluorine, chlorine, bromine, iodine, etc. Among others, fluorine and chlorine are preferable.
Examples of the alkyl in the optionally substituted alkyl as the substituents for R1 include a straight or branched C1-10 alkyl such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, etc., and preferably lower (C1-6) alkyl.
Examples of the substituents in the optionally substituted alkyl include halogen (e.g. fluorine, chlorine, bromine, iodine, etc.), nitro, cyano, hydroxy group, thiol group, amino group, carboxyl group, an optionally halogenated C1-4 alkoxy (e.g. methoxy, ethoxy, trifluoromethoxy, trifluoroethoxy, etc.), C2-4 alkanoyl (e.g. acetyl, propionyl, etc.), C1-4 alkylsulfonyl (e.g. methanesulfonyl, ethanesulfonyl, etc.), etc., and the number of the substituents are preferably 1 to 3.
Examples of the cycloalkyl in the optionally substituted cycloalkyl as the substituents for R1 include C3-7 cycloalkyl, etc. such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, etc.
Examples of the substituents in the optionally substituted cycloalkyl include halogen (e.g. fluorine, chlorine, bromine, iodine, etc.), nitro, cyano, hydroxy group, thiol group, amino group, carboxyl group, an optionally halogenated C1-4 alkyl (e.g. trifluoromethyl, methyl, ethyl, etc.), an optionally halogenated C1-4 alkoxy (e.g. methoxy, ethoxy, trifluoromethoxy, trifluoroethoxy, etc.), C2-4 alkanoyl (e.g. acetyl, propionyl, etc.), C1-4 alkylsulfonyl (e.g. methanesulfonyl, ethanesulfonyl, etc.), etc., and the number of the substituents are preferably 1 to 3.
Examples of the substituents in the optionally substituted hydroxy group as the substituents for R1 include
(1) an optionally substituted alkyl (e.g. C1-10 alkyl such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl,.etc., preferably lower (C1-6) alkyl, etc.);
(2) an optionally substituted cycloalkyl (e.g. C3-7 cycloalkyl, etc. such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, etc.);
(3) an optionally substituted alkenyl (e.g. C2-10 alkenyl such as allyl, crotyl, 2-pentenyl, 3-hexenyl, etc., preferably lower (C2-6)alkenyl, etc.);
(4) an optionally substituted cycloalkenyl (e.g. C3-7 cycloalkenyl, etc. such as 2-cyclopentenyl, 2-cyclohexenyl, 2-cyclopentenylmethyl, 2-cyclohexenylmethyl, etc.);
(5) an optionally substituted aralkyl (e.g. phenyl-C1-4 alkyl (e.g. benzyl, phenethyl, etc.), etc.);
(6) an optionally substituted acyl (e.g. C2-4 alkanoyl (e.g. acetyl, propionyl, butyryl, isobutyryl, etc.), C1-4 alkylsulfonyl (e.g. methanesulfonyl, ethanesulfonyl, etc.), etc.);
(7) an optionally substituted aryl (e.g. phenyl, naphthyl, etc.); etc.
Examples of the substituents which the above-mentioned (1) optionally substituted alkyl, (2) optionally substituted cycloalkyl, (3) optionally substituted alkenyl, (4) optionally substituted cycloalkenyl, (5) optionally substituted aralkyl, (6) optionally substituted acyl and (7) optionally substituted aryl may have include halogen (e.g. fluorine, chlorine, bromine, iodine, etc.), nitro, cyano, hydroxy group, thiol group, amino group, carboxyl group, an optionally halogenated C1-4 alkyl (e.g. trifluoromethyl, methyl, ethyl, etc.), an optionally halogenated C1-4 alkoxy (e.g. methoxy, ethoxy, trifluoromethoxy, trifluoroethoxy, etc.), C2-4 alkanoyl (e.g. acetyl, propionyl, etc.), C1-4 alkylsulfonyl (e.g. methanesulfonyl, ethanesulfonyl, etc.), etc., and the number of the substituents are preferably 1 to 3.
Examples of the substituents in the optionally substituted thiol group as the substituents for R1 are the same as the above-described substituents in the optionally substituted hydroxy group as the substituents for R1, and among others,
(1) an optionally substituted alkyl (e.g. C1-10 alkyl such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, etc., preferably lower (C1-6) alkyl, etc.);
(2) an optionally substituted cycloalkyl (e.g. C3-7 cycloalkyl, etc. such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, etc.);
(3) an optionally substituted aralkyl (e.g. phenyl-C1-4 alkyl (e.g. benzyl, phenethyl, etc.), etc.);
(4) an optionally substituted aryl (e.g. phenyl, naphthyl, etc.); etc. are preferable.
Examples of the substituents which the above-mentioned (1) optionally substituted alkyl, (2) optionally substituted cycloalkyl, (3) optionally substituted aralkyl and (4) optionally substituted aryl may have include halogen (e.g. fluorine, chlorine, bromine, iodine, etc.), nitro, cyano, hydroxy group, thiol group, amino group, carboxyl group, an optionally halogenated C1-4 alkyl (e.g. trifluoromethyl, methyl, ethyl, etc.), an optionally halogenated C1-4 alkoxy (e.g. methoxy, ethoxy, trifluoromethoxy, trifluoroethoxy, etc.), C2-4 alkanoyl (e.g. acetyl, propionyl, etc.), C1-4 alkylsulfonyl (e.g. methanesulfonyl, ethanesulfonyl, etc.), etc., and the number of the substituents are preferably 1 to 3.
Examples of the substituents in the optionally substituted amino group as the substituents for R1 are the same as the above-described substituents in the optionally substituted hydroxy group as the substituents for R1, and examples of the optionally substituted amino group as the substituents for R1 include an amino group which may have one to two substituents selected from the above-described substituents in the optionally substituted hydroxy group as the substituents for R1, etc. Among others, as the substituents in the optionally substituted amino group as the substituents for R1,
(1) an optionally substituted alkyl (e.g. C1-10 alkyl such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, etc., preferably lower (C1-6) alkyl, etc.);
(2) an optionally substituted cycloalkyl (e.g. C3-7 cycloalkyl, etc. such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, etc.);
(3) an optionally substituted alkenyl (e.g. C2-10 alkenyl such as allyl, crotyl, 2-pentenyl, 3-hexenyl, etc., preferably lower (C2-6) alkenyl, etc.);
(4) an optionally substituted cycloalkenyl (e.g. C3-7 cycloalkenyl, etc. such as 2-cyclopentenyl, 2-cyclohexenyl, 2-cyclopentenylmethyl, 2-cyclohexenylmethyl, etc.);
(5) an optionally substituted acyl (e.g. C2-4 alkanoyl (e.g. acetyl, propionyl, butyryl, isobutyryl, etc.), C1-4 alkylsulfonyl (e.g. methanesulfonyl, ethanesulfonyl, etc.), etc.);
(6) an optionally substituted aryl (e.g. phenyl, naphthyl, etc.); etc. are preferable.
Examples of the substituents, which each of the above-described (1) optionally substituted alkyl, (2) optionally substituted cycloalkyl, (3) optionally substituted alkenyl, (4) optionally substituted cycloalkenyl, (5) optionally substituted acyl and (6) optionally substituted aryl may have, include halogen (e.g. fluorine, chlorine, bromine, iodine, etc.), nitro, cyano, hydroxy group, thiol group, amino group, carboxyl group, an optionally halogenated C1-4 alkyl (e.g. trifluoromethyl, methyl, ethyl, etc.), an optionally halogenated C1-4 alkoxy (e.g. methoxy, ethoxy, trifluoromethoxy, trifluoroethoxy, etc.), C2-4 alkanoyl (e.g. acetyl, propionyl, etc.), C1-4 alkylsulfonyl (e.g. methanesulfonyl, ethanesulfonyl, etc.), etc., and the number of the substituents are preferably 1 to 3.
The substituents in the optionally substituted amino group as the substituents for R1 may bind to each other to form a cyclic amino group (e.g. 5- to 6-membered cyclic amino, etc. such as pyrrolidine, pyrroline, piperazine, piperidine, morpholine, thiomorpholine, pyrrole, imidazole, etc.). Said cyclic amino group may have a substituent, and examples of the substituents include halogen (e.g. fluorine, chlorine, bromine, iodine, etc.), nitro, cyano, hydroxy group, thiol group, amino group, carboxyl group, an optionally halogenated C1-4 alkyl (e.g. trifluoromethyl, methyl, ethyl, etc.), an optionally halogenated C1-4 alkoxy (e.g. methoxy, ethoxy, trifluoromethoxy, trifluoroethoxy, etc.), C2-4 alkanoyl (e.g. acetyl, propionyl, etc.), C1-4 alkylsulfonyl (e.g. methanesulfonyl, ethanesulfonyl, etc.), etc., and the number of the substituents are preferably 1 to 3.
Examples of the optionally substituted acyl as the substituents for R1 include a carbonyl group or a sulfonyl group binding to
(1) hydrogen;
(2) an optionally substituted alkyl (e.g. C1-10 alkyl such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, etc., preferably lower (C1-6) alkyl, etc.);
(3) an optionally substituted cycloalkyl (e.g. C3-7 cycloalkyl, etc. such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, etc.);
(4) an optionally substituted alkenyl (e.g. C2-10 alkenyl such as allyl, crotyl, 2-pentenyl,3-hexenyl, etc., preferably lower (C2-6) alkenyl, etc.);
(5) an optionally substituted cycloalkenyl (e.g. C3-7 cycloalkenyl, etc. such as2-cyclopentenyl, 2-cyclohexenyl, 2-cyclopentenylmethyl, 2-cyclohexenylmethyl, etc.);
(6) an optionally substituted 5- to 6-membered monocyclic aromatic group (e.g. phenyl, pyridyl, etc.); etc.
Examples of the acyl include acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, heptanoyl, octanoyl, cyclobutanecarbonyl, cyclopentanecarbonyl, cyclohexanecarbonyl, cycloheptanecarbonyl, crotonyl, 2-cyclohexenecarbonyl, benzoyl, nicotinoyl, methanesulfonyl, ethanesulfonyl, etc.
Examples of the substituents, which the above-mentioned (2) optionally substituted alkyl, (3) optionally substituted cycloalkyl, (4) optionally substituted alkenyl, (5) optionally substituted cycloalkenyl and (6) optionally substituted 5- to 6-membered monocyclic aromatic group may have, include halogen (e.g. fluorine, chlorine, bromine, iodine, etc.), nitro, cyano, hydroxy group, thiol group, amino group, carboxyl group, an optionally halogenated C1-4 alkyl (e.g. trifluoromethyl, methyl, ethyl, etc.), an optionally halogenated C1-4 alkoxy (e.g. methoxy, ethoxy, trifluoromethoxy, trifluoroethoxy, etc.), C2-4 alkanoyl (e.g. acetyl, propionyl, etc.), C1-4 alkylsulfonyl (e.g. methanesulfonyl, ethanesulfonyl, etc.), etc., and the number of the substituents are preferably 1 to 3.
Examples of the optionally esterified carboxyl group as the substituents for R1 include a carbonyloxy group binding to
(1) hydrogen;
(2) an optionally substituted alkyl (e.g. C1-10 alkyl such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, etc., preferably lower (C1-6) alkyl, etc.);
(3) an optionally substituted cycloalkyl (e.g. C3-7 cycloalkyl, etc. such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, etc.);
(4) an optionally substituted alkenyl (e.g. C2-10 alkenyl such as allyl, crcotyl, 2-pentenyl,3-hexenyl, etc., preferably lower (C2-6) alkenyl, etc.);
(5) an optionally substituted cycloalkenyl (e.g. C3-7 cycloalkenyl, etc. such as 2-cyclopentenyl, 2-cyclohexenyl, 2-cyclopentenylmethyl, 2-cyclohexenylmethyl, etc.);
(6) an optionally substituted aryl (e.g. phenyl, naphthyl, etc.); etc., and preferably carboxyl, lower (C1-6) alkoxy-carbonyl, aryloxycarbonyl (e.g. methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, phenoxycarbonyl, naphthoxycarbonyl, etc.), etc.
Examples of the substituents, which the above-mentioned (2) optionally substituted alkyl, (3) optionally substituted cycloalkyl, (4) optionally substituted alkenyl, (5) optionally substituted cycloalkenyl and (6) optionally substituted aryl may have, include halogen (e.g. fluorine, chlorine, bromine, iodine, etc.), nitro, cyano, hydroxy group, thiol group, amino group, carboxyl group, an optionally halogenated C1-4 alkyl (e.g. trifluoromethyl, methyl, ethyl, etc.), an optionally halogenated C1-4 alkoxy (e.g. methoxy, ethoxy, trifluoromethoxy, trifluoroethoxy, etc.), C2-4 alkanoyl (e.g. acetyl, propionyl, etc.), C1-4 alkylsulfonyl (e.g. methanesulfonyl, ethanesulfonyl, etc.), etc., and the number of the substituents are preferably 1 to 3.
Examples of the aromatic group in the optionally substituted aromatic group as the substituents for R1 include 5- to 6-membered homocyclic or heterocyclic ring aromatic ring, etc. such as phenyl, pyridyl, furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, isothiazolyl, isoxazolyl, tetrazolyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazolyl, etc.
Examples of the substituents for these aromatic group include halogen (e.g. fluorine, chlorine, bromine, iodine, etc.), nitro, cyano, hydroxy group, thiol group, amino group, carboxyl group, an optionally halogenated C1-4 alkyl (e.g. trifluoromethyl, methyl, ethyl, etc.), an optionally halogenated C1-4 alkoxy (e.g. methoxy, ethoxy, trifluoromethoxy, trifluoroethoxy, etc.), C2-4 alkanoyl (e.g. acetyl, propionyl, etc.), C1-4 alkylsulfonyl (e.g. methanesulfonyl, ethanesulfonyl, etc.), etc., and the number of the substituents are preferably 1 to 3.
The number of the above-mentioned substituents for R1 is 1-4 (preferably 1-2) and they may be same or different and present at any possible position on the ring represented by R1. When two or more substituents are present on the xe2x80x9cphenyl groupxe2x80x9d and the xe2x80x9cthienyl groupxe2x80x9d of the xe2x80x9coptionally substituted phenyl groupxe2x80x9d and xe2x80x9coptionally substituted thienyl groupxe2x80x9d represented by R1, two substituents among them may bind to each other to form a lower (C1-6) alkylene (e.g. trimethylene, tetramethylene, etc.), a lower (C1-6) alkyleneoxy (e.g. xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94CH2xe2x80x94CH2xe2x80x94, etc.), a lower (C1-6) alkylenedioxy (e.g. xe2x80x94Oxe2x80x94CH2xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94CH2xe2x80x94CH2xe2x80x94Oxe2x80x94, etc.), a lower (C2-6) alkenylene (e.g. xe2x80x94CH, xe2x80x94CHxe2x95x90CHxe2x80x94, xe2x80x94CH2xe2x80x94CH2xe2x80x94CHxe2x95x90CHxe2x80x94, xe2x80x94CH2xe2x80x94CHxe2x95x90CHxe2x80x94CH2xe2x80x94, etc.), a lower (C4-6) alkadienylene (e.g. xe2x80x94CHxe2x95x90CHxe2x80x94CHxe2x95x90CHxe2x80x94, etc.), etc.
Preferred examples of the xe2x80x9csubstituentsxe2x80x9d, which the xe2x80x9cphenyl groupxe2x80x9d and the xe2x80x9cthienyl groupxe2x80x9d of the xe2x80x9coptionally substituted phenyl groupxe2x80x9d and xe2x80x9coptionally substituted thienyl groupxe2x80x9d represented by R1 may have, include an optionally halogenated lower (C1-4) alkyl (e.g. methyl, ethyl, t-butyl, trifluoromethyl, etc.), an optionally halogenated lower (C1-4) alkoxy (e.g. methoxy, ethoxy, t-butoxy, trifluoromethoxy, etc.), halogen (e.g. fluorine, chlorine, etc.), nitro, cyano, an amino group optionally substituted with 1-2 lower (C1-4) alkyl groups (e.g. amino, methylamino, dimethylamino, etc.), 5- to 6-membered cyclic amino (e.g. 1-pyrrolidinyl, 1-piperazinyl, 1-piperidinyl, 4-morpholino, 4-thiomorpholino, 1-imidazolyl, 4-tetrahydropyranyl, etc.), etc., and when R1 is a phenyl group, the xe2x80x9csubstituentxe2x80x9d is preferably present at para position.
In the above formula (I), Y is xe2x80x94CH2xe2x80x94, xe2x80x94Sxe2x80x94 or xe2x80x94Oxe2x80x94; and preferably xe2x80x94CH2xe2x80x94 or xe2x80x94Oxe2x80x94.
In the above formula (I), R2, R3and R4 are independently an optionally substituted aliphatic hydrocarbon group or an optionally substituted alicyclic heterocyclic ring group.
Examples of the xe2x80x9caliphatic hydrocarbon groupxe2x80x9d in the xe2x80x9coptionally substituted aliphatic hydrocarbon groupxe2x80x9d represented by R2, R3 and R4 include
(1) an optionally substituted alkyl (e.g. C1-10 alkyl such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, etc., preferably lower (C1-1) alkyl, etc.);
(2) an optionally substituted cycloalkyl (e.g. C3-8, cycloalkyl, etc. such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.); provided that
(3) an optionally substituted alkenyl (e.g. C2-10 alkenyl such as allyl, crotyl, 2-pentenyl, 3-hexenyl, etc., preferably lower (C2-6)alkenyl, etc.);
(4) an optionally substituted cycloalkenyl (e.g. C3-7 cycloalkenyl, etc. such as 2-cyclopentenyl, 2-cyclohexenyl, 2-cyclopentenylmethyl, 2-cyclohexenylmethyl, etc.); etc.
Examples of the xe2x80x9calicyclic heterocyclic ring groupxe2x80x9d in the xe2x80x9coptionally substituted alicyclic heterocyclic ring groupxe2x80x9d represented by R2, R3 and R4 include a 5- to 6-membered non-aromatic heterocyclic ring containing 1 to 4 hetero-atoms consisting of 1 to 2 kinds of hetero-atoms selected from oxygen atom, sulfur atom and nitrogen atom such as tetrahydrofuran, tetrahydrothiophene, dithiolane, oxathiolane, pyrrolidine, pyrroline, imidazolidine, imidazoline, pyrazolidine, pyrazoline, piperidine, piperazine, oxazine, oxadiazine, thiazine, thiadiazine, morpholine, thiomorpholine, pyran, tetrahydropyran, etc.; preferably a saturated 5- to 6-membered heterocyclic ring, etc., such as tetrahydrofuran; piperidine, tetrahydropyran, tetrahydrothiopyran, etc.); etc.
Examples of the substituents, which the xe2x80x9caliphatic hydrocarbon groupxe2x80x9d and the xe2x80x9calicyclic heterocyclic ring groupxe2x80x9d in the xe2x80x9coptionally substituted aliphatic hydrocarbon groupxe2x80x9d and the xe2x80x9coptionally substituted alicyclic heterocyclic ring groupxe2x80x9d represented by R2, R3 and R4 may have, include halogen (e.g. fluorine, chlorine, bromine, iodine, etc.), an optionally halogenated lower (C1-4) alkyl, an optionally halogenated C1-4 alkoxy (e.g. methoxy, ethoxy, trifluoromethoxy, trifluoroethoxy, etc.), C2-4 alkanoyl (e.g. acetyl, propionyl, etc.), C1-4 alkylsulfonyl (e.g. methanesulfonyl, ethanesulfonyl, etc.), phenyl, phenyl-lower (C1-4) alkyl, C3-7 cycloalkyl, cyano, nitro, oxo, hydroxy group, thiol group, amino group, carboxyl group, lower (C1-4) alkoxy-carbonyl (preferably, halogen, an optionally halogenated lower (C1-4) alkyl, an optionally halogenated lower (C1-4) alkoxy, phenyl-lower (C1-4) alkyl, C3-7 cycloalkyl, cyano, oxo, hydroxy group, etc.), etc., and the number of the substituents are preferably 1 to 3.
In the above formula (I), as the group R2 or R3, an optionally substituted acyclic hydrocarbon group is preferable, an optionally substituted alkyl group is more preferable. In particular, the groups R2 and R3 are preferably the same and more preferably both of the groups R2 and R3 are methyl.
In the above formula (I), as the group R4, an optionally substituted alicyclic hydrocarbon group or an optionally substituted alicyclic heterocyclic ring group and more preferably an optionally substituted cycloalkyl group or an optionally substituted saturated alicyclic heterocyclic ring group. In particular, R4 is preferably an optionally substituted cyclohexyl or an optionally substituted 6-membered saturated alicyclic heterocyclic ring group and more preferably an optionally substituted cycloalkyl, an optionally substituted tetrahydropyranyl, an optionally substituted tetrahydrothiopyranyl or an optionally substituted piperidyl.
The compound of the formula (I) of the present invention may be hydrated or solvated. When the compound of the formula (I) of the present invention exists as configuration isomer, diastereomer, conformer, etc., it is possible to isolate individual isomers with per se known separation and purification method, if desired. When the compound of the formula (I) of the present invention is racemate, it can be separated into (S)-compound and (R)-compound with usual optical resolution and individual optical isomers and a mixture thereof are included in the scope of the present invention.
The pro-drug of Compound (I) means a compound which is converted to Compound (I) under the physiological condition or with a reaction due to an enzyme, an gastric acid, etc. in the living body, that is, a compound which is converted to Compound (I) with oxidation, reduction, hydrolysis, etc. according to an enzyme; a compound which is converted to Compound (I) with gastric acid, etc.; etc.
Examples of the pro-drug of Compound (I) include a compound wherein an amino group of Compound (I) is substituted with acyl, alkyl, phosphoric acid, etc. (e.g. a compound wherein an amino group of Compound (I) is substituted with eicosanoyl, alanyl, pentylaminocarbonyl, (5-methyl-2-oxo-1,3-dioxolen-4-yl)methoxycarbonyl, tetrahydrofuranyl, pyrrolidylmethyl, pivaloyloxymethyl, tert-butyl, etc.); a compound wherein an hydroxy group of Compound (I) is substituted with acyl, alkyl, phosphoric acid, boric acid, etc. (e.g. a compound wherein an hydroxy group of Compound (I) is substituted with acetyl, palmitoyl, propanoyl, pivaloyl, succinyl, fumaryl, alanyl, dimethylaminomethylcarbonyl, etc.); a compound wherein a carboxyl group of Compound (I) is modified with ester, amide, etc. (e.g. a compound wherein a carboxyl group of Compound (I) is modified with ethyl ester, phenyl ester, carboxymethyl ester, dimethylaminomethyl ester, pivaloyloxyrmethyl ester, ethoxycarbonyloxyethyl ester, phthalidyl ester, (5-methyl-2-oxo-1,3-dioxolen-4-yl)methyl ester, cyclohexyloxycarbonylethyl ester, methyl amide, etc.); etc. These pro-drug can be produced by per se known method from Compound (I).
The pro-drug of Compound (I) may be a compound which is converted into Compound (I) under the physiological conditions as described in xe2x80x9cPharmaceutical Research and Developmentxe2x80x9d, Vol. 7 (Drug Design), pages 163-198 published in 1990 by Hirokawa Publishing Co. (Tokyo, Japan).
Compound (I) may be labeled with isotope (e.g. 3H, 14C, 35S, 125I, etc.), etc.
The present compound of the formula (I) alone or as an admixture with a pharmaceutically acceptable carrier may be non-orally, administered as a liquid preparation such as an injection.
Examples of the carriers include various organic or inorganic carriers which are generally used in this field. For example, a solvent, a solubilizer, a suspending agent, a isotonizing agent, a buffer, a soothing agent, etc. are used in the liquid formulations. In addition, if desired, an appropriate additive such as a preservative, an antioxidant, a colorant, a sweetener, etc. may be used in the above formulations.
Examples of the solvent include water for injection, alcohol, propyleneglycol, macrogol, sesame oil, corn oil, etc. Examples of the solubilizer include polyethyleneglycol, propyleneglycol, polyoxyethylene (20) sorbitan mono-oleate (polysorbate 80), D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, etc. Examples of the suspending agent include surfactants such as stearyl triethanolamine, sodium laurylsulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzetonium chloride, glycerin monostearate, etc.; hydrophilic polymers such as polyvinylalcohol, polyvinylpyrrolidone, sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, etc.; etc. Examples of the isotonizing agent include sodium chloride, glycerin, D-mannitol, etc. Examples of the buffer include a buffer solution of phosphate, acetate, carbonate, citrate, etc. Examples of the soothing agent include benzylalcohol, etc. Examples of the preservative include paraoxybenzoic acid esters, chlorobutanol, benzylalcohol, phenethylalcohol, dehydroacetic acid, sorbic acid, etc. Examples of the antioxidant include sulfites, ascorbic acid, etc.
The compound of the formula (I) of the present invention may be used in combination with other drug for the treatment or prevention of infectious disease of HIV (in particular, a pharmaceutical composition for the treatment or prevention of AIDS). In this case, these drugs can be formulated by mixing individually or simultaneously with pharmaceutically acceptable carriers, excipients, binders, diluents or the like, which can be administered non-orally as a pharmaceutical composition for the treatment or prevention of infectious disease of HIV. In the case of formulating these effective components individually, while the individually formulated agents can be administered in the form of their mixture prepared by using e.g. a diluent when administered, the individually formulated agents can also be administered separately or simultaneously or with time intervals to the one and same subject. A kit for a administering the individually formulated effective components in the form of their mixture prepared by using e.g. a diluent when administered (e.g. a kit for injection which comprises two or more ampoules each comprising a powdery component and a diluent for mixing and dissolving two or more components when administered, etc.), etc. are also included by the pharmaceutical composition of the present invention.
Example of the other pharmaceutical agent for the treatment or prevention of infectious disease of HIV to be used in combination with the compound of the formula (I) of the present invention include nucleoside reverse transcriptases inhibitor such as zidovudine, didanosine, zalcitabine, lamivudine, stavudine, abacavir, adefovir, adefovir dipivoxil, fozivudine tidoxil, etc.; non-nucleoside reverse transcriptases inhibitor (including an agent having anti-oxidation activity such as immunocal, oltipraz, etc.) such as nevirapine, delavirdine, efavirenz, loviride, immunocal, oltipraz, etc.; protease inhibitors such as saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, palinavir, lasinavir, etc.; etc.
As the nucleoside reverse transcriptase inhibitor, zidovudine, didanosine, zalcitabine, lamivudine, stavudine, etc. are preferable; as the non-nucleoside reverse transcriptase inhibitor, nevirapine, delavirdine, etc. are preferable; and as the protease inhibitor, saquinavir, ritonavir, indinavir, nelfinavir, etc. are preferable.
The compound of the formula (I) can be produced in accordance with per se known methods, for example, the methods described below, the methods described in JP-A-73476/1996, or analogous methods thereto.
A salt of the compound of the formulas (I), (II), (III), (IV), (V), (I-1), (I-2) and (I-3) may form a salt with inorganic base, organic base, inorganic acid, organic acid, basic or acidic amino acid, etc., as long as such a salt does not interfere the reaction. Examples of the salt with the inorganic base include a salt with alkali metal (e.g. sodium, potassium, etc.), alkaline earth metal (e.g. calcium, magnesium, etc.), aluminum, ammonium, etc. Examples of the salt with the organic base include a salt with trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, N,Nxe2x80x2-dibenzylethylenediamine, etc. Examples of the salt with the inorganic acid include a salt with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc. Examples of the salt with the organic acid include a salt with formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. Examples of the salt with the basic amino acid include a salt with arginine, lysine, ornithine, etc. Examples of the salt with the acidic amino acid include a salt with aspartic acid, glutamic acid, etc.
In the following reaction steps, when the starting compounds have, as substituents, an amino group, a carboxyl group and/or hydroxy group, these groups may be protected by ordinary protective groups such as those generally employed in peptide chemistry, etc. After the reaction, if necessary, the protective groups may be removed to obtain the desired compound.
Examples of the amino-protective group include an optionally substituted formyl, an optionally substituted C1-6 alkyl-carbonyl (e.g. methylcarbonyl, ethylcarbonyl, etc.), phenylcarbonyl, C1-6 alkyloxy-carbonyl (e.g. methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl, etc.), aryloxycarbonyl (e.g. phenoxycarbonyl, etc.), C7-10 aralkyloxy-carbonyl (e.g. benzyloxycarbonyl, etc.), trityl, phthaloyl, etc. These protective groups may be substituted by 1 to 3 substituents such as halogen atom (e.g. fluorine, chlorine, bromine, iodine, etc.), C1-6 alkyl-carbonyl (e.g. acetyl, propionyl, butyryl, etc.), nitro group, etc.
Examples of the carboxyl-protective group include an optionally substituted C1-6 alkyl (e.g. methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, etc.), phenyl, trityl, silyl, etc. These protective groups may be substituted by 1 to 3 substituents such as halogen atom (e.g. fluorine, chlorine, bromine, iodine, etc.), formyl, C1-6 alkyl-carbonyl (e.g. acetyl, propionyl, butyryl, etc.), nitro group, etc.
Examples of the hydroxy-protective group include an optionally substituted C1-6 alkyl (e.g. methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, etc.), phenyl, C7-10 aralkyl (e.g. benzyl, etc.), formyl, C1-6 alkyl-carbonyl (e.g. acetyl, propionyl, etc.), phenyloxycarbonyl, C7-10 aralkyloxycarbonyl (e.g. benzyloxycarbonyl, etc.), pyranyl, furanyl, silyl, etc. These protective groups may be substituted by 1 to 4 substituents such as halogen atom (e.g. fluorine, chlorine, bromine, iodine, etc.), C1-6 alkyl, phenyl, C7-10 aralkyl, nitro group, etc.
These protective group may be introduced or removed by per se known methods (e.g. a method described in Protective Groups in Organic Chemistry (J. F. W. McOmie et al.; Plenum Press Inc.) or the methods analogous thereto. For example, employable method for removing the protective groups is a method using an acid, a base, reduction, ultraviolet ray, hydrazine, phenylhydrazine, sodium N-methyldithiocarbamate, tetrabutylammonium fluoride, palladium acetate, etc. 
wherein Xxe2x88x92 is an counter anion (e.g. an anion of a halogen atom (e.g. Clxe2x88x92, Brxe2x88x92, Ixe2x88x92, etc.), etc.) and the other symbols are as defined above.
This production method is carried out by reacting the compound (II) with the aniline derivative (III) to obtain the anilide Compound (I-1).
The condensation reaction of the compounds (II) and (III) is carried out by usual methods for peptide synthesis. Said methods for peptide synthesis are employed according to optional known methods, for example, methods described in xe2x80x9cPeptide Synthesisxe2x80x9d written by M. Bodansky and M. A. Ondetti, Interscience, New York, 1966; xe2x80x9cThe Proteinsxe2x80x9d, volume 2, written by F. M. Finn and K. Hofmann, H. Nenrath and R. L. Hill edition, Academic Press Inc., New York, 1976; xe2x80x9cpeputido-gosei no kiso to jikken (Basis and Experiment of Peptide Synthesis)xe2x80x9d written by Nobuo Izumiya et al., Maruzen K. K. ,1985; etc., as well as azide method, chloride method, acid anhydride method, mixed acid anhydride method, DCC method, active ester method, method using Woodward reagent K, carbonyldiimidazole method, oxidation-reduction method, DCC/HONB method, etc. and in addition WSC method, method using diethyl cyanophosphate (DEPC), etc.
The condensation reaction can be carried out in a solvent. Examples of the solvents to be employed in the reaction include anhydrous or hydrous N,N-dimethylformamide (DMF), dimethylsulfoxide, pyridine, chloroform, dichloromethane, tetrahydrofuran, dioxane, acetonitrile, or a suitable mixture of these solvents. The reaction temperature is generally about xe2x88x9220xc2x0 C. to about 50xc2x0 C., preferably about xe2x88x9210xc2x0 C. to about 30xc2x0 C. and the reaction time is generally about 1 to about 100 hours, preferably about 2 to about 40 hours. 
When Compound (I-2) has a tertiary amine residue, Compound (I-1) having an quaternary ammonium can be produced by reacting Compound (I-2) with halogenated alkyl. Examples of a halogen atom include chlorine, bromine, iodine, etc. and usually about 1 to 5 moles of the halogenated alkyl (e.g. halogenated lower (C1-6) alkyl, etc. ) is used per mole of Compound. (I-2). The reaction is carried out in an inert solvent such as toluene, benzene, xylene, dichloromethane, chloroform, 1,2-dichloroethane, dimethylformamide, dimethylaceatamide, etc., or a suitable mixture of these solvents. The reaction temperature is generally about 10xc2x0 C. to about 160xc2x0 C., preferably about 20xc2x0 C. to about 120xc2x0 C. and the reaction time is generally about 1 hour to about 100 hours, preferably about 2 hours to about 40 hours. This reaction is preferably carried out under inert gas (e.g. nitrogen, argon, etc.) atmosphere. 
wherein V in the Compound (IV) is a halogen atom (chlorine, bromine, iodine, etc.), or a sulfonyloxy group (methane-sulfonyloxy group, trifluoromethanesulfonyloxy group, benzenesulfonyloxy group, toluenesulfonyloxy group, etc.), and the other symbols are as defined above.
Compound (I-1) having a quaternary ammonium can be produced by reacting Compound (IV) and a tertiary amine. The reaction is carried out in an inert solvent such as toluene, benzene, xylene, dichloromethane, chloroform, 1,2-dichloroethane, dimethylformamide (DMF), dimethylacetamide, etc., or a suitable mixture of these solvents. Usually, about 1-3 moles of the tertiary amine is used per mole of Compound (IV). The reaction temperature is generally about 10xc2x0 C. to about 120xc2x0 C., and the reaction time is generally about 1 hour to about 40 hours. This reaction is preferably carried out under inert gas (e.g. nitrogen, argon, etc.) atmosphere. 
Compound (I-1) can be produced by subjecting Compound (V) wherein Vxe2x80x2 is a halogen atom (bromine, iodine, etc.) or a sulfonyloxy group (trifluoromethanesulfonyloxy group, etc.), and the other symbols are as defined above to, for example, Suzuki reaction [cross condensation reaction of aryl borate, with e.g. aryl halide or aryloxytrifluoromethanesulfonate in the presence of palladium catalyst; A. Suzuki et al., Synth. Commun. 1981, 11, 5131]. Usually, about 1-1.5 times moles of aryl borate is used per mole of Compound (V).
The thus obtained anilide derivative (I-1) can be isolated and purified by known separation and purification methods such as concentration, concentration under reduced pressure, extraction, crystallization, recrystallization, solvent convert, chromatography, etc.
If necessary, the thus obtained compound (I-1) can be converted into the desired compound (I) wherein X is chlorine by per se known method (e.g. a method using ion exchange resin, exchange reaction in a solvent containing an excess amount of sodium chloride, etc.).
Compound (II) used as a starting material can be produced by a known method (e.g. method described in JP-A-73476/1996, etc.) or the methods analogous thereto. For example, Compound (II) can be produced by a method described in the following Reaction Scheme I, a method described in the following Reference Examples or the methods analogous thereto. 
wherein R9 is a C1-4 alkyl group, and the other symbols are as defined above.
In this reaction, the compound of the formula (VI) is heated with a polyphosphoric acid, or Compound (VI) is converted to acid chloride with thionyl chloride, oxalyl chloride, phosphorous oxychloride, phosphorous pentachloride, etc., followed by subjecting the resulting acid chloride to usual Friedel-Crafts reaction and cyclizing the same to produce Compound (VII). Compound (VII) is reacted with carbonate ester in the presence of a base to produce ketoester (VIII). Compound (VIII) is subjected to reduction with catalytic hydrogenation or sodium boron hydride, etc. to produce Compound (IX). Compound (IX) is subjected to dehydration and ester hydrolysis by per se known methods to produce unsaturated carboxylic acid (II).
Compound (III) can be produced by a known method (e.g. method described in JP-A-73476/1996, etc.) or the methods analogous thereto. For example, Compound (III) can be produced by a method described in the following Reaction Scheme II, a method described in the following Reference Examples or the methods analogous thereto. 
The reduction of Compound (X) can be carried out by per se known methods, for example, reduction with metal, reduction with metal hydride, reduction with metal hydride complex compound, reduction with diborane or substituted borane, catalytic hydrogenation, etc. That is, this reaction is carried out by treating Compound (X) with reduction agent. Examples of the reduction agent include metal such as reduced iron, zinc powder, etc.; alkali metal boron hydride (e.g. sodium boron hydride, lithium boron hydride, etc.); metal hydride complex compound such as aluminum lithium hydride, etc.; metal hydride such as sodium hydride etc.; organic tin compound (triphenyltin hydride, etc.), metal complex compound and metal salt such as nickel compound, zinc compound etc.; catalytic reduction agent using hydrogen and transit metal catalyst such as palladium, plutinum, rhodium, etc.; diborane; etc. Among others, as the reduction agent, catalytic reduction agent using hydrogen and transit metal catalyst such as palladium,plutinum, rhodium, etc.; reduced iron, etc. are preferable. The reaction is carried out in a solvent which does not inhibit the reaction. Examples of the solvent include benzene, toluene, xylene, chloroform, carbon tetrachloride, dichloromethane, 1,2-dichloroethane, 1,1,2,2-tetrachloroethane, diethylether, tetrahydrofuran, dioxane, methanol, ethanol, propanol, isopropanol, 2-methoxyethanol, N,N-dimethylformamide, acetic acid, or a suitable mixture of these solvents, etc. The solvent is appropriately selected depending on kind of the reduction agent. The reaction temperature is generally about xe2x88x9220xc2x0 C. to about 150xc2x0 C., preferably about 0xc2x0 C. to about 100xc2x0 C., and the reaction time is generally about 1 to about 24 hours.
The resulting Compound,(III) can be separated and purified with know separation and purification methods such as concentration, concentration under reduced pressure, extraction, crystallization, was recrystallized with, solvent conversion, chromatography, etc.
The compound of the formula (I) of the present invention has potent CCR5 antagonistic activity and therefore can be used for the treatment or prevention of various infectious diseases of HIV, for example, AIDS in human. The compound of the formula (I) of the present invention is low toxic and safely used as CCR5 antagonist for the treatment or prevention of AIDS and also for the prevention of the progression of AIDS. The compound of the formula (I) of the present invention has superior solubility and absorbability, and therefore it can be advantageously used as injection.
The dose per day of the compound of the formula (I) varies depending on the condition and body weight of a patient, administration route, etc. Typical daily dose per adult patient (body weight: 50 Kg) for subcutaneous administration is about 1-200 mg, preferably about 1-100 mg, more preferably about 2-50 mg, and in particular about 5-30 mg, as active ingredient [the compound of the formula (I)] and the compound of the formula (I) is administered once or 2-3 times par day.
When the compound of the formula (I) is used in combination with a reverse transcriptase inhibitor and/or a protease inhibitor, the dose of the reverse transcriptase inhibitor or the protease inhibitor ranges, for example, from about 1,/200-1/2 or more of usual dose to about 2-3 times or less of usual dose. In case that two or more drugs are used in combination, each dose of the drugs is appropriately adjusted if one drug affects metabolism of the other drug, while each dose of the drugs when they are used in combination is generally the same as the dose when they are used alone.
Typical daily dose of the reverse transcriptase inhibitor and the protease inhibitor is as follows:
zidovudine: 100 mg
didanosine: 125-200 mg
zalcitabine: 0.75 mg
lamivudine: 150 mg
stavudine: 30-40 mg
saquinavir: 600 mg
ritonavi: 600 mg
indinavir: 800 mg
nelfinavir: 750 mg
In case of combination use of the compound of the formula (I) with a reverse transcriptase inhibitor and/or a protease inhibitor preferred embodiments are shown below.
{circle around (1)} A drug containing about 1-200 mg of the compound of the formula (I) and a drug containing about 50-200 mg of zidovudine to one adult patient (body weight: 50 Kg) are administered. Each of the drugs may be administered to the one and the same subject simultaneously or with time intervals of 12 hours or less.
{circle around (2)} A drug containing about 1-200 mg of the compound of the formula (I) and a drug containing about 300-1200 mg of saquinavir to one adult patient (body weight: 50 Kg) are administered. Each of the drugs may be administered to the one and the same subject simultaneously or with time intervals of 12 hours or less.
The present invention is hereinafter described in more detail by means of the following Test Example, Reference Example and Working Example, which are mere examples of the present invention and are not construed as limitative to the present invention.
The following gene manipulation is carried out in accordance with methods described in textbook (Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, 1989) or protocol attached to reagents.